B. Protein Blotting

A general protocol for sample preparation.

Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.

Wash membrane again four times for 5 min each in TBST.

The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

Source / Purification

Background

RANTES/CCL5 (regulated upon activation, T cell expressed and secreted) is a member of the "C-C" or β family of chemokines that induce inflammation and are associated with a number of inflammatory disorders (1,2). RANTES is produced and secreted mainly by CD8+ T cells, macrophages, and platelets, as well as epithelial cells, fibroblasts and some solid tumors (2-7). RANTES acts as a chemoattractant and has other regulatory functions on a number of cell types including monocytes, memory T cells, NK cells, eosinophils, basophils, dendritic cells, and mast cells (3, 7-9). Signaling by RANTES is mediated by several G-protein coupled receptors (GPCRs), including CCR1, CCR3, CCR4 and CCR5.